Analyte sensing mediated by adapter/carrier molecules

ABSTRACT

This invention relates to an improved method and system for sensing of one or more analytes. A host molecule, which serves as an adapter/carrier, is used to facilitate interaction between the analyte and the sensor element. A detectable signal is produced reflecting the identity and concentration of analyte present.

CROSS REFERENCE TO RELATED APPLICATIONS

This is a divisional of U.S. application Ser. No. 09/441,376 filed Nov.17, 1999, by Hagan Bayley, Orit Braha and LiQun Gu and entitled “AnalyteSensing Mediated by Adapter/Carrier Molecules,” now U.S. Pat. No.6,426,231 dated Jul. 30, 2002.

This application claims the benefit of U.S. Provisional PatentApplication No. 60/109,034 filed Nov. 18, 1998.

RIGHTS IN THE INVENTION

This invention was made in part with United States Government supportunder grants from DARPA, DOE, ONR, and the United States Government hascertain rights in the invention.

BACKGROUND

1. Field of the Invention

This invention relates to the field of biosensing and, moreparticularly, to an improved method and system for analyte sensingmediated by adapter/carrier molecules which act as an adapter betweenthe analyte and sensor element or deliver the analyte to the sensorelement.

2. Description of the Background

Stochastic sensing is based on the detection of individual bindingevents between analyte molecules and a single receptor, which acts as abiosensor element. The read-out depends on a property of the receptor,usually a protein, that is altered when the binding site is occupied. Inits simplest manifestation, stochastic sensing provides a binary signal(occupied/unoccupied) comprising fluctuations in, e.g. electricalcurrent, fluorescence, or force. The frequency of occurrence of thebinding events is determined by the concentration of the analyte. Thenature of the binding events, e.g. the magnitude and duration of theassociated signal, is determined by the properties of the analyte. Theability to identify an analyte by its characteristic signature is adistinctive feature of stochastic sensing.

The ability to observe changes in the state of single protein moleculeshas been available with respect to ion channels for over twenty years.The electrical currents generated by the large ion fluxes through thesemolecules (e.g., 10⁸ s⁻¹) can be monitored by single channel recording.More recently, structural changes in single protein molecules have beendetected by fluorescence techniques and by force measurements (Doleman,B. J., et al., Proc. Natl. Acad. Sci. USA 95:5442-5447, 1998; Hellinga,H. W., et al., Trends Biotechnol. 16:183-189, 1998; Czarnik, A. W.,Nature 394:417-418, 1998).

Genetically engineered versions of the bacterial pore forming proteinα-Hemolysin (αHL) have been used as sensor elements. Current stochasticschemes are limited in that the type of analytes that can be sensed arerestricted to those which interact with the pore. Current schemes cannotbe used, for example, to analyze organic molecules, molecules insolublein aqueous media and certain mixtures of analytes.

Better sensors would be useful in many situations. For example, inmedicine, improved means for detecting physiological markers andtherapeutic agents are needed; in environmental protection, variouspollutants and effluents from factories must be monitored morethoroughly; for defense, new ways to detect explosives and chemical andbiological agents are urgently required. In only a few cases are theavailable technologies optimal for the task at hand. Better devices areneeded with improved sensitivity and rapid “real time” response.

There is therefore a need for analyte sensors, including stochasticbiosensors, that can detect the presence and concentration of a widervariety of analytes as well as samples containing mixtures of analytes.

SUMMARY OF THE INVENTION

The present invention overcomes the problems and disadvantagesassociated with current strategies and designs and provides analytesensing systems and methods which extend the categories of analytes thatgive a response and improve the dynamic range of response. A hostmolecule is used to serve as an adapter between the analyte and sensorelement, or to deliver the analyte directly to the sensor element,producing a unique signal that indicates both the concentration andidentity of the analyte. The invention is particularly useful indetecting organic molecules.

The present invention is a substantial improvement over stochasticsensing using pore proteins as sensor elements without the benefit of ahost molecule. The host molecule of the present invention allowsinteractions between an analyte and a sensor element that would notnormally occur. The host molecule of the present invention canconcentrate analyte, even from the vapor phase. The host molecule maymediate the analysis of organic molecules that are normally insoluble inaqueous media. Two or more analyte molecules that interact with a hostmolecule can be analyzed simultaneously with a single sensor element.The complex signal can be resolved to reveal the concentrations ofmultiple components in a mixture. In addition, more than one hostmolecule can be used in combination with a single sensor element.

Accordingly, one embodiment of the invention is directed to a system forsensing at least one analyte comprising a sensor element and a hostmolecule. The sensor element has a receptor site. The host molecule,which acts as a carrier or adapter, is configured to interact with boththe receptor site of the sensor element and the analyte to produce adetectable signal unique for the analyte.

Sensing may comprise identifying or quantitating/determining theconcentration of the analyte, or both. The host molecule is preferably acyclodextrin, such as β-cyclodextrin (βCD), and the sensor element ispreferably an α-Hemolysin (αHL) pore.

Systems of the invention may be highly combinatorial. For example,another embodiment of the invention is directed to a system for sensinga plurality of different analytes comprising a plurality of differentsensor elements, each sensor element comprising a pore and having areceptor site, and a plurality of different host molecules. The hostmolecules are each configured to interact with a receptor site of one ofthe plurality of different sensor elements and one of the differentanalytes to produce a detectable and unique signal.

Another embodiment of the invention is directed to a biosensor fordetecting an analyte in a sample comprising a bilayer, which separatesthe biosensor into a first compartment and a second compartment, and asensor element disposed in the first compartment so that it forms achannel in the bilayer. The biosensor further comprises a host moleculewhich is designed or configured to interact with a receptor site on thesensor element and the analyte to produce a detectable signal. The hostmolecule may be disposed in the first, second or both compartments.

Another embodiment of the invention is directed to a method for sensingat least one analyte in a sample comprising the steps of providing abiosensor, the biosensor comprising a sensor element having a receptorsite and a host molecule, the host molecule configured to interact withthe receptor site of the sensor element and the analyte to produce adetectable signal, allowing the sample to interact with the biosensor toproduce a signal, and detecting the signal.

Still another embodiment of the invention is directed to a method ofmaking a biosensor for detecting an analyte in a sample comprisingproviding a bilayer apparatus, the bilayer apparatus comprising abilayer separating the apparatus into a first compartment and a secondcompartment, adding a sensor element to the first compartment andallowing it to form a channel in the bilayer, and providing a hostmolecule, the host molecule being configured to interact with a receptorsite on the sensor element and the analyte to produce a detectablesignal. The method further comprises the step of adding the hostmolecule to the first or second compartments or both. The host moleculemay be added substantially simultaneously with the addition of thesample.

Another embodiment is directed to a method for modifying an interactiveproperty of a protein with a second molecule comprising modifying theinteractive property of the protein by contacting the protein with athird molecule, said third molecule comprising a non-covalent molecularadapter.

Other embodiments and advantages of the invention are set forth in partin the description which follows, and in part, will be obvious from thisdescription, or may be learned from the practice of the invention.

DESCRIPTION OF THE DRAWINGS

FIGS. 1 a-d are bilayer recordings showing the interaction of a singleαHL pore with βCD and the model analytes 2-adamantanamine (A₁) and1-adamantanecarboxylic acid (A₂).

FIG. 2 is a diagram illustrating the operation of the system accordingto a preferred embodiment of the present invention.

FIG. 3 a is a molecular graphics representation of the interactionbetween αHL and βCD depicting a sagittal section through the pore withβCD lodged in the lumen of the transmembrane channel.

FIG. 3 b is a molecular graphics representation of the interactionbetween αHL and βCD depicting a view into the channel from the transside with βCD bound.

FIG. 3 c is a molecular graphics representation of the interactionbetween αHL and βCD depicting a view into the channel from the transside with βCD removed.

FIG. 4 a is a diagram illustrating the response of αHL●βCD at differentanalyte concentrations.

FIG. 4 b are plots of P_(P●C●A)/P_(P●C) versus [A] for 2-adamantanamineand 1-adamantanecarboxylic acid.

FIG. 4 c are plots of [C●A] versus [C]●[A] for 2-adamantanamine and1-adamantanecarboxylic acid.

FIG. 5 a is a diagram illustrating analysis of currents from binarysolutions of analytes showing current amplitude histograms for a singleαHL pore in the presence of βCD and 2-adamantanamine (A₁) and1-adamantanecarboxylic acid (A₂).

FIG. 5 b illustrates the experimentally measured concentrations of A₁and A₂, determined from the data in 4 a, plotted against the actualconcentration of A₁.

FIG. 6 is a diagram illustrating analyte identification data acquiredfrom operation of the system of the present invention.

FIG. 7 a is a sagittal section through a WT (wild-type) αHL pore.

FIG. 7 b shows the structures of the β-cyclodextrins used in Examples9-11.

FIG. 7 c is a schematic of the WT-αHL pore showing βCD lodged in thelumen of the channel.

FIG. 7 d shows sequences of the transmembrane β barrels in WT-αHL,(left) and αHL-CH1 (right).

FIG. 8 a is a representative bilayer current recording through theWT-αHL pore by cyclodextrin adapters in the presence of 40 μM βCD addedto the trans side of the bilayer.

FIG. 8 b is a representative bilayer current recording through theWT-αHL pore by cyclodextrin adapters in the presence of 40 μM s₇βCDadded to the trans side.

FIG. 8 c depicts I-V curves showing modulation of single channelcurrents through the WT-αHL pore by cyclodextrin adapters, specificallyI-V curves for αHL (□), βHL•βCD (◯) and αHL•s₇βCD (●) based onrecordings made with cis: 1000 mM KCl; trans, 200 mM KCl.

FIG. 8 d depicts I-V curves showing modulation of single channelcurrents through the WT-αHL pore by cyclodextrin adapters, specificallyI-V curves for αHL (□), αHL•βCD (◯), and αHL•s₇βCD (●), based onrecordings made with cis: 200 mM KCl; trans, 1000 mM KCl.

FIG. 8 e depicts I-V curves showing modulation of single channelcurrents through the WT-αHL pore by cyclodextrin adapters, specificallyI-V curves under biionic conditions in 10 mM phosphate buffer, pH 7.5: □and ▪, αHL and αHL•s₇βCD with cis: 1000 mM KCl; trans, 1000 mM NaCl; ◯and ●, αHL and αHL•s₇βCD with cis: 1000 mM NaCl; trans, 1000 mM KCl.

FIG. 9 a are bilayer current recordings showing modulation of singlechannel currents through the αHL-CH1 pore by βCD in the presence of 40μM βCD added to the trans side of the bilayer.

FIG. 9 b depicts I-V curves showing modulation of single channelcurrents through the αHL-CH1 pore by βCD, specifically I-V curves forβHL-CH1 (□) and αHL-CH1•βCD (◯) based on recordings made with cis: 1000mM KCl; trans, 100 mM KCl.

FIG. 10 is a summary of charge selectivity data.

DESCRIPTION OF THE INVENTION

As embodied and broadly described herein, the present invention isdirected to an improved method and system for analyte sensing mediatedby carrier/adapter molecules. A host molecule is used to provide abinding site in the sensor element, thereby acting as an adapter betweenthe sensor element and analyte, or to deliver the analyte directly tothe sensor element. In either case, a unique signal is produced thatindicates both the concentration and identity of the analyte.

In the adapter mode, the host molecule actually changes the bindingproperties of the sensor element, such as a protein, allowing it tointeract with an analyte, such as an organic molecule, therebyfacilitating detection of the analyte.

The present invention combines single molecule detection with proteinengineering. Protein engineering allows the generation of an enormousvariety of analyte binding sites in receptor macromolecules. Geneticengineering may be by design based on structural data, as well as bydirected evolution by random or semi-random mutagenesis and geneshuffling. It also includes targeted chemical modification of proteins,enhanced by cutting-edge techniques in organic synthesis includingcombinatorial approaches.

Engineered transmembrane protein pores are useful sensor elements forstochastic detection (Braha, O., et al., Chem. Biol. 4:497-505, 1997).In their simplest manifestation, they produce a binary signal(occupied/unoccupied) comprising fluctuations in electrical current. Theconcentration of the analyte determines the frequency of occurrence ofthe fluctuations and, in a distinctive feature of stochastic sensing,the identity of the analyte is revealed by the “signature” of thebinding events, e.g., the magnitude and duration of the currentfluctuations.

Genetically engineered versions of the bacterial pore-forming proteinα-hemolysin (αHL) have been used to identify and quantitate divalentmetal ions in solution (Braha, O., et al., Chem. Biol. 4:497-505, 1997;also see U.S. patent application Ser. No. 09/122,583 filed Jul. 24,1997, incorporated herein by reference). In one such study, the analytebinding site was placed in the lumen of the transmembrane channel.However, in this model it is likely that electrostatic effects, ratherthan steric block or a conformational change in the protein, werelargely responsible for the modulation of current by analyte. As such,this strategy is of limited value in the detection of organic molecules,which may not necessarily be charged.

In contrast, the present invention allows for the detection ofnon-charged organic molecules. Analyte sensing using αHL equipped with anon-covalent molecular adapter according to the invention can beeffectively used to distinguish and quantitate a variety of molecules,including organic molecules. Further, a single sensor element can beused to analyze mixtures of the analytes.

α-Hemolysin is an exotoxin secreted by Staphylococcus aureus (Gouaux,E., J. Struct. Biol. 121:110-122, 1998). The monomeric 293 amino acidpolypeptide can self assemble on lipid bilayers to form a heptamericpore. The pore self assembles efficiently into bilayers either throughthe monomer (Menestrina, G., J. Membrane Biol., 90:177-190, 1986) or asa preformed heptamer (Braha, O., et al., Chem. Biol., 4:497-505, 1997).The pore is a mushroom-shaped structure in which the lower half of thestem, a 14-stranded β barrel, forms a transmembrane channel (Song, L.,et al., Science, 274:1859-1865, 1996). Transported molecules movethrough a 100 Å-long channel centered on the molecular 7-fold axis(Song, L., et al., Science 274:1859-1865, 1996). The opening of thechannel on the cis side of the bilayer (the side of assembly) is ˜76 Åabove the membrane surface and 29 Å in diameter. The channel widens intoa roughly spherical vestibule ˜42 Å in diameter. About 20 Å above themembrane plane the vestibule narrows and becomes a 14-stranded β barrel,52 Å in length, that continues through the membrane averaging about 20 Åin diameter. The trans entrance to the channel lies close to the bilayersurface. Roughly globular molecules of up to ˜2000 Da (Krasilnikov, O.V., et al., FEMS Microbiol. Immunol. 105:93-100, 1992; Bezrukov, S. M.et al., Macromolecules 29:8517-8522, 1996) orelongated polymers ofhigher mass, such as single stranded nucleic acids (Kasianowicz, J. J.,et al., Proc. Natl. Acad. Sci. USA 93:13770-13773, 1996) can passthrough the αHL pore. The protein is robust; for example, the heptameris stable at up to 65° C.

It was observed that α-, β- and γ-cyclodextrins (CD) at micromolarconcentrations enter the channel and produce reversible partial blocksof the ionic current. In view of this observation, βCD (M_(τ)=1135 Da)was examined further. The results of this examination are described morefully in Example 3, below. The block was established when βCD was addedfrom the trans side of a planar bilayer (FIGS. 1 a, b) but not from thecis side. The kinetics of the block were consistent with a simple schemein which there is a single binding site for βCD within the lumen of thechannel for which k^(C) _(on)=5.46×10⁴ M⁻¹s⁻¹, k^(C) _(off)=1.15×10²s⁻¹, and K^(C) _(ƒ)=4.75×10² M⁻¹ in 1 M NaCl, 10 mM Na phosphate and 5μM EDTA at pH3.0 (trans). The conductance of αHL was 750 pS (SD=21,n=35) in the absence of βCD and 272 pS (SD=9, n=35) in the presence ofβCD.

Because the block was partial (64%) and because cyclodextrins are knownto act as hosts for a variety of guest molecules, it was postulated thatguests might produce further reductions in single channel currents.Indeed, this has been discovered to be the case, as demonstrated inExample 3, below. Specifically, 2-adamantanamine (A₁, 80 μM trans)reduced the conductance of the partially blocked channel to 126.5 pS(SD=0.5, n=7) with a residence time (τ_(A1)) of 2.54 msec (SD=0.21), buthad no effect on the completely open channel (FIG. 1 c). A second guest,1-adamantanecarboxylic acid (A₂, 20 μM trans), also reduced theconductance of the partially blocked channel, this time to 112.2 pS(SD-3.2, n=7) with a residence time (τ_(A2)) of 14.0 msec (SD=0.8). Theguests competed for the single binding site in the αHL●βCD complex, sothat events due to each could be monitored in a mixture (FIG. 1 d).These observations suggested a kinetic scheme and model for theinteractions of αHL, βCD and the analytes (FIG. 3).

The kinetic scheme (Scheme 1) suggested by the observations is asfollows:

Scheme 1

where: P, αHL pore; C, the adapter βCD; A_(i), guests analytes; C●A,P●C, P●C●A_(i), the various non-covalent complexes of P, C and A_(i).

FIG. 2 is a schematic that further illustrates the operation of thesystem of the present invention. Referring to FIG. 2, the forward andbackward rate constants for steps 1, 2 and 3 can be measured by singlechannel recording. The recording trace reveals both the concentrationand identity of the analyte.

More specifically, the host-guest chemistry of the invention involvestwo mechanisms. In one mechanism, shown in steps 2 and 4 of FIG. 2, thehost-guest chemistry uses a “carrier” mechanism whereby the host(carrier) delivers the guest (analyte) to the pore. Alternately, in the“adapter” mechanism shown in FIG. 2, steps 1 and 3, the host (adapter)is lodged in the pore while the guest (analyte) associates anddisassociates. Where the residence time of the analyte within thecyclodextrin host is relatively long, the host acts as a carrier ratherthan an adapter.

Channel modulation occurs only in the presence of the carrier/adapter(host), e.g., cyclodextrin. Detailed studies support the interpretationof FIG. 2. Depending on the analyte and host, transitions associatedwith 1 and 2, or with 3, can dominate the signal. In either case, asignal that can be interpreted in terms of the concentration andidentity of the analyte is obtained. Importantly, the analyte canmodulate the single channel conductance while the host is bound and/ormodulate the kinetics of the interaction of the host with the channel.

FIGS. 3 a-c are molecular graphics diagrams of the model for theinteractions of αHL, βCD and the analytes. Specifically, FIGS. 3 a-c arethree views of a molecular graphics representation of the interactionbetween αHL and βCD. The structures of αHL were generated from thecoordinates (Song, L., et al., Science 274:1859-1865, 1996) by usingSPOCK 6.3 (Christopher, SPOCK: the structural properties observation andcalculation kit (program manual), Center for Macromolecular Design,Texas A&M University, College Station, Tex., 1998). The βCD structurewas generated with Insight II 97.0. The two structures were scaled andsuperimposed in Adobe Photoshop 4.0. FIG. 3 a is a sagittal section viewthrough the heptameric αHL pore showing βCD lodged in the lumen of thetransmembrane channel. FIG. 3 b is a view into the channel from thetrans side with βCD bound. FIG. 3 c is a view into the channel from thetrans side, βCD removed.

As shown in FIGS. 3 a-c, βCD fits snugly into the lumen of the αHLchannel with its molecular 7-fold axis parallel to the 7-fold axis ofthe heptameric pore. A guest molecule, such as an adamantane derivative,can in turn fit within the βCD cavity. Thus, βCD acts as a molecularadapter in a stochastic sensing system where αHL is the sensor elementand guest molecules are analytes. The kinetic scheme explains theobserved interactions of analytes in simple solutions or in mixtures. Inthe case of βCD and the adamantane derivatives described here, theresidence time of the cyclodextrin in the lumen of the pore isrelatively long compared to the residence time of the analyte in thecyclodextrin and βCD acts as an adapter. As noted, where the residencetime of the analyte within the cyclodextrin host is relatively long, thehost acts as a carrier rather than an adapter.

As described below in Example 5, in the practice of the invention, thesignal from an analyte can be used not only to identify the analyte butalso to quantitate it. As expected, the frequency of αHL●βCD occupancyby analyte increases with analyte concentration (FIG. 4 a).Interestingly, the residence time of βCD●2-adamantanamine on αHL isgreater than βCD itself (FIG. 4 a), which is true for all βCD●A in thisstudy.

For a single analyte, Scheme 1 gives:P _(P●C●A) /P _(P●C) =K ^(A) _(ƒ)[A](see Eq. 10 in Example 7, below) where P_(P●C●A) and P_(P●C) are theexperimentally determined probabilities of occurrence of the states ofthe αHL pore with both βCD and analyte bound or with only βCD bound,K^(A) _(ƒ) is the equilibrium formation constant for αHL●βCD●A fromanalyte (A) and αHL●βCD, and [A] is the concentration of free analyte.[A] can be determined from the experimental values of P_(P●C),P_(P)(P_(P) is the probability of occurrence of the unoccupied αHLpore), [A]₀ and [C]₀ (the total concentrations of analyte and βCD) andK^(C) _(ƒ) (the equilibrium formation constant for the αHL●βCD complex),which can be measured separately (see Example 7). Therefore, K^(A) _(ƒ)values can be obtained from the slope of a linear plot (FIG. 4 b). Inthis way, K^(A) _(ƒ) for 1-adamantanecarboxylic acid and2-adamantanamine (at pH 3.0) were found to be respectively1.35±0.19×10⁵M⁻¹ and 1.03±0.15×10⁴M⁻¹ (mean±SD, n=7), corresponding toΔG values of −7.0 kcal mol⁻¹ and −5.5 kcal mol⁻¹.

Values of K^(A′) _(ƒ), the equilibrium formation constant for theanalyte (A) with βCD in solution can be obtained from plots of:[C●A]=K ^(A′) _(ƒ) [C][A](see Eq. 11 in Example 7, below) where values for [C●A], [C] and [A],the concentrations of the βCD●A complex, free βCD and free analyterespectively, can be obtained from experimental or known values ofP_(P●C), P_(P), [A]₀, [C]₀ and K^(C) _(ƒ) (see Example 7). K^(A) _(ƒ)values at pH 3.0 for 1-adamantanecarboxylic acid and 2-adamantanaminewere found to be respectively 5.76±1.50×10⁴ M⁻¹ and 9.84±2.19×10³ M⁻¹(mean±SD, n=7), corresponding to AG values of −6.5 kcal mol⁻¹ and −5.5kcal mol⁻¹, which are closely similar to the values with βCD bound tothe αHL pore. Literature AG values for 1-adamantanecarboxylic acidbinding to βCD, determined by NMR in solution, are in the same range asthose determined herein at −7.5 kcal mol⁻¹ (pH 4.05) and −6.4 kcal mol⁻¹(pH 7.2) (Rekharsky, M. V., et al., Chem. Rev. 98:1875-1917, 1998).

In a working sensor, the total concentration [A_(i)]₀ of analyte A_(i)of known K^(Ai) _(ƒ) and K^(Ai′) _(ƒ) would be determined by thefollowing equation (see Eq. 8 in Example 7, below):$\left\lbrack A_{i} \right\rbrack_{0} = {\frac{1}{K_{f}^{A_{i}}} \cdot \left( {\frac{1}{P_{P \cdot C}} + \frac{K_{f}^{A_{i}^{\prime}}}{P_{P} \cdot K_{f}^{C}}} \right) \cdot P_{P \cdot C \cdot A_{i}}}$

Another important attribute of sensing according to the invention isthat two or more analytes, only one of which can occupy the receptor ata given moment, can be identified and quantitated “simultaneously” by asingle sensor element. In a mixture, signals from different analytes arerecognized by their characteristic extents of channel block andresidence times. To illustrate this with αHL and the βCD adapter, anexperiment was performed in which 1-adamantanecarboxylic acid (A₂) waskept constant at 20 μM, while 2-adamantanamine (A₁) was varied. Theresults are shown below in Example 4. Current amplitude histograms (FIG.5 a), which revealed P_(P), P_(P●C) and P_(P●C●Ai), and Eq. 8 were usedto generate the total concentrations of the two analytes, [A₁]₀ and[A₂]₀. The values obtained were in close agreement with the actualconcentrations in the mixtures (FIG. 5 b).

Analyte sensing with adapter molecules provides a highly versatileapproach for analyte identification and quantitation. The concept bearssome resemblance to olfaction in which carrier molecules (olfactorybinding proteins) deliver odorants to membrane-bound receptors(Bianchet, M. A., et al., Nature Struct. Biol. 3:934-939, 1996). As innature, both the adapter/carrier and the receptor may be varied. Forexample, the range of analytes that can be detected may be extended byusing additional naturally occurring and chemically modifiedcyclodextrins (Rekharsky, M. V., et al., Chem. Rev. 98:1875-1917, 1998)as adapters. Additional host molecules that have been developed bysynthetic chemists may be considered as adapter/carriers (Chen, H., etal., Curr. Op. Chem. Biol. 1:458-466, 1997; Arduini, A., et al., Curr.Op. Chem. Biol. 1:467-474, 1997; Beer, P. D., et al., Curr. Op. Chem.Biol. 1:475-482, 1997). When necessary, genetically engineered αHL pores(Bayley, H., Sci. Am. 277(3):62-67, 1997) or pores other than αHL(Hartgerink, J., D., et al., Chem. Eur. J. 4:1367-1372, 1998; Schmid,B., et al., Protein Sci. 7:1603-1611, 1998) may be used to accommodatethe alternative adapters, which, as discussed below, may be covalentlyattached to the pores. Such adapters may covalently attach at the mouthor within the interior of a pore.

In addition to changes in electrical current, various modes ofdetection, such as those amenable to stochastic sensing with singlemolecule sensor elements, e.g., fluorescence and force measurements, maybe used in the practice of the invention. (Weiss, S., Science,283:1676-1683, 1999; Xie, X. S., et al., J. Biol. Chem.,274:15967-15970, 1999). For example, single molecule fluorescence (Xie,X. S., Acc. Chem. Res. 29:598-606, 1996) or force detection methods(Oberhauser, A. F., et al., Nature 393:181-185, 1998) may be consideredfor read-out.

For applications in the field, a rugged version of stochastic sensorelements may be used as was recently achieved for a sensor based onmacroscopic channel currents (Cornell, B. A., et al., Nature387:580-583, 1997). Thus, the present invention is not limited to thelaboratory bilayer systems as described in the Examples, but alsoencompasses sensor elements incorporated into more rugged devices foruse in the field.

The present invention is a substantial improvement over stochasticsensing using pore proteins as sensor elements without the benefit of anadapter/carrier. The adapter/carrier of the present invention allowsinteractions between an analyte and a sensor element that would notnormally occur (for example, adamantane-1-carboxylic acid itself doesnot bind to the pore and therefore produces no signal). Theadapter/carrier of the present invention can concentrate analyte, evenfrom the vapor phase. The adapter/carrier may mediate the analysis oforganic molecules that are normally insoluble in aqueous media. Two ormore analyte molecules that interact with an adapter/carrier can beanalyzed simultaneously with a single sensor element. The complex signalcan be resolved to reveal the concentrations of multiple components in amixture. In addition, more than one adapter/carrier can be used incombination with a single sensor element.

Analyte sensing using the novel systems of the present inventionprovides a number of advantages. These include: high sensitivity; rapidresponse (milliseconds to seconds in the nanomolar concentration range);reversibility; wide dynamic range; both the concentration and identityof an analyte are determined; the sensor element need not be highlyselective—each analyte produces a characteristic signal; severalanalytes can be quantitated concurrently by a single sensor element;lack of simultaneous competition by similar analytes at the singlebinding site; fouling cannot give a false reading—signal is notcharacteristic of an analyte; no loss of signal-to-noise at low analyteconcentrations; digital output for facile electronic interfacing; andself-calibration and reagentless operation.

As noted, the use of modified cyclodextrins extends the categories ofanalytes that give a response and also improves the dynamic range of theresponse. In addition to natural, modified and synthetic cyclodextrins,many different classes of host molecules may be used. The use ofgenetically engineered pores to produce new interactions ofadapters/carriers with pores, to better accommodate variousadapters/carriers, and to alter the kinetics of existing interactionscan further extend the categories of analytes that can be examined andthe dynamic range of the response.

For instance, host molecules other than cyclodextrins, includingnaturally occurring, synthetic and genetically engineered materials(e.g., peptide polymers, synthetic host molecules, etc.) may be usedwithout departing from the spirit and scope of the invention. Inaddition to non-covalent adapters (e.g., cyclodextrins and relatedmolecules), adapters may also be covalently attached to the proteins orpores. For example, single poly(ethylene glycol) molecules (PEG's) havebeen covalently attached to the interior of the αHL pore. Thus, theinvention is not limited to cyclodextrins; rather, a wide range ofresponsive molecules can be used as adapters for sensing purposes,including but not limited to, PEG's (including derivatized PEG's),synthetic polymers other than PEG, oligonucleotides, aptamers, peptidepolymers, oligosaccharides, etc.

As with non-covalent adapters/carriers, mutant αHLs may be used tobetter accommodate the covalent adapters. In addition to αHL, other poreproteins can be used. As with non-covalent adapters, the invention canalso be used with alternative sensor elements including enzymes,antibodies, receptors, etc. Therefore, as with non-covalentadapters/carriers, the invention is highly combinatorial.

Analyte sensing according to the invention using host molecules (e.g.,both covalent and non-covalent adapters) and sensor elements (e.g.,protein pores) is highly combinatorial with respect to analytes,adapters and the proteins that accommodate them. A vast number of host(adapter/carrier) molecules can be used in combination with a vastnumber of different pores, genetically engineered to accommodate thehost molecule. The invention effectively provides a huge tool box ofpossible adapters and proteins which can be mixed and matched to make avariety of different biosensors. These different biosensor elements canthen be combined as desired into an array made up of multiple differentbiosensors.

Further, analyte delivery according to the invention is not limited tostochastic sensing, but may be applied in other detection modes as well.For example, it can be used in biosensors that rely on multiple channelsin a membrane, i.e., it can be used in both the multichannel(macroscopic current) mode and for stochastic sensing.

A wide range of analytes of broad interest can be examined by theadapter approach of the invention. For example, a variety of therapeuticdrugs can be recognized and their concentrations determined (Gu, L.-Q.,et al., Nature, 398:686-690, 1999). Further, cyclodextrins and adaptersother than β-cyclodextrin are effective, expanding the range of analytesthat can be examined. Remarkably, αHL can be engineered to better securethe adapters. For example, homoheptameric pores of the mutant αHL-M113Nbind β-cyclodextrin ˜10⁴ times more tightly than WT homoheptamers (L.-Q.Gu and S. Cheley, unpublished).

With respect to the lipid bilayers used in the practice of theinvention, a more stable environment for lipid bilayers may be providedby solid supports (Heyse, S., et al., Biochim. Biophys. Acta,1376:319-338, 1998; Sackmann, E., Science, 271:43-48, 1996). Recent workhas focused on making defect-free supported bilayers with an aqueouslayer between the bilayer and the support to facilitate theincorporation of pores and provide a reservoir of electrolyte (Heyse,S., et al., Biochim. Biophys. Acta, 1376:319-338, 1998). Two groups havereported the switching of protein pores in bilayers supported on goldsurfaces (Cornell, B. A., et al., Nature, 387:580-583, 1997; Stora, T.,et al., Angew. Chem. Int. Ed. Engl., 38:389-392, 1999).

Less conventional approaches for accommodating functional protein poresmay prove useful. Planar bilayers can be strengthened by the depositionof S layers (the two-dimensional porous crystalline arrays of a singleprotein that envelop many bacterial species) and remain able toincorporate active αHL pores (Schuster, B., et al., Biochim. Biophys.Acta, 1370:280-288, 1998). Active engineered pores maybe placed innanoscale apertures, which can be produced in a variety of materials(Hulteen, J. C., et al., J. Am. Chem. Soc., 120:6603-6604, 1998; Sun,L., et al., Langmuir, 15:738-741, 1999). The chemistry of the reoipientsurface could be tailored to make it more compatible with the proteinand the protein may be reciprocally engineered.

Accordingly, one embodiment of the invention is directed to a system forsensing at least one analyte in a sample comprising a sensor element anda host molecule. The sensor element has a receptor site. The hostmolecule, which acts as an adapter or carrier, is configured to interactwith both the receptor site of the sensor element and the analyte toproduce a detectable signal. When the host molecule functions as anadapter between the analyte and the receptor site, the host molecule maybe covalently or non-covalently attached to the receptor site. Thesensing may comprise stochastic sensing.

The sensor element may be disposed in a membrane, such as a bilayer. Forexample, the system may further comprise a bilayer and the sensorelement comprises a channel disposed in the bilayer.

In one such embodiment, the system further comprises a bilayerapparatus. The bilayer apparatus may assume any suitable configuration,including rugged devices for use in the field or laboratory devices. Thebilayer apparatus comprises a bilayer separating the bilayer apparatusinto a first compartment and a second compartment. The sensor element isdisposed in the bilayer so that it forms a channel in the bilayer. Forexample, the sensor element may be disposed in either the first orsecond or both compartments, i.e., by stirring, so that it forms achannel in the bilayer. The host molecule is disposed (or may bedisposed during actual testing for the analyte) in either compartment orboth compartments, as required by the application.

For example, referring to FIG. 7 c, sensor element 2 forms a pore 4 inbilayer 6 creating a cis side or compartment 8 and a trans side orcompartment 10. Host molecule 12 is lodged in pore 4.

As will be clear to those of skill in the art, the operation of thesystem may be varied without departing from the spirit and scope of theinvention. For instance, the sensor element may disposed in the first orsecond compartment so that it forms a channel in the bilayer and thehost molecule may be disposed in the second compartment, the firstcompartment, or both compartments. In other words, for those adaptersthat attach non-covalently, the host molecule may be delivered to eitheror both mouths of the pore (not just the trans side). Likewise, analytesmay be delivered to either mouth.

As will further be clear to those of skill in the art, with respect tocovalent adapters, in a prefered embodiment, the adapter is attached tothe protein (i.e., αHL) before or after assembly of the pore by targetedchemical modification.

Sensing may comprise identifying or quantitating/determining theconcentration of the analyte, or both. The host molecule may be anatural, synthetic or modified cyclodextrin, such as βCD or s₇βCD. Othersuitable hosts which adapt to the receptor site of the sensor elementmay also be used, including, but not limited to, poly(ethylene glycol)molecules (including derivatized PEG's), synthetic polymers other thanPEG, oligonucleotides, aptamers, peptide polymers and oligosaccharides.

The sensor element may be a protein, such as a transmembrane pore,enzyme, antibody or receptor. In a preferred embodiment, the sensorelement comprises or functions as a pore, such as a geneticallyengineered transmembrane protein pore. Preferred sensor elements includeα-Hemolysin (αHL) pores, including wild-type α-Hemolysin (αHL) pores andgenetically engineered or mutant α-Hemolysin (αHL) pores.

The signal produced by the analyte may be a change in electricalcurrent, such as a change in the magnitude of the current. The signalmay comprise the duration of the change in the current. Alternately, thesignal may be a change in fluorescence, a change in force, or otherunique signal.

The system may be used to sense more than one analyte. Analytes whichmay be detected include, but are not limited to, organic molecules andmolecules lacking a charge.

Another embodiment of the invention is directed to a system for sensinga plurality of different analytes comprising a plurality of differentsensor elements, each sensor element comprising a pore and having areceptor site, and a plurality of different host molecules, the hostmolecules each configured to interact with a receptor site of one of theplurality of different sensor elements and one of the different analytesto produce a detectable signal.

Another embodiment of the invention is directed to a biosensor fordetecting an analyte in a sample comprising a bilayer which separatesthe biosensor into a first compartment and a second compartment, asensor element disposed in the bilayer so that it forms a channel in thebilayer, and a host molecule. The host molecule is configured tointeract with a receptor site on the sensor element and the analyte toproduce a detectable signal.

Preferably, the sensor element is disposed in the first compartment andstirred so that it forms a channel in the bilayer, and the host moleculeis disposed in either the first or second compartment, or both. In apreferred embodiment, the host molecule is disposed in the secondcompartment during testing for the analyte, i.e., the host molecule andsample are added to the second compartment substantially simultaneously.

Another embodiment of the invention is directed to a method for sensingat least one analyte in a sample comprising providing a biosensor, thebiosensor comprising a sensor element having a receptor site and a hostmolecule, the host molecule configured to interact with the receptorsite of the sensor element and the analyte to produce a detectablesignal, allowing the sample to interact with the biosensor to produce asignal, and detecting the signal. Preferably, sensing comprisesstochastic sensing. The host molecule may function as an adapter orcarrier.

The biosensor may further comprise a bilayer and the sensor elementcomprises a channel disposed in the bilayer. In one embodiment, thesystem further comprises a bilayer apparatus which comprises a bilayerseparating the apparatus into a first compartment or side and a secondcompartment or side and the sensor element is disposed in the bilayer sothat it forms a channel in the bilayer.

In a preferred method, the sensor element is added to the firstcompartment and stirred so that it forms a channel in the bilayer andthe step of allowing the sample to interact with the biosensor comprisesadding the host molecule and the analyte to the first compartment, thesecond compartment, or both compartments.

Still another embodiment is directed to a method of making a biosensorfor detecting an analyte in a sample comprising providing a bilayerapparatus, the bilayer apparatus comprising a bilayer separating thebilayer apparatus into a first compartment and a second compartment,adding a sensor element to the first compartment and stirring, therebyallowing the sensor element to form a channel in the bilayer, andproviding a host molecule. The host molecule is configured to interactwith a receptor site on the sensor element and the analyte to produce adetectable signal. The method may further comprise the step of addingthe host molecule to the first or second compartment, or both. In apreferred embodiment, the host molecule is added to the secondcompartment substantially simultaneously with the addition of thesample.

Another embodiment is directed to a method for modifying an interactiveproperty of a protein with a second molecule (i.e., how the protein andsecond molecule interact with each other, if at all). This methodcomprises the step of modifying the interactive property of the proteinby contacting the protein with a third molecule, the third moleculecomprising a non-covalent molecular adapter.

In addition to the foregoing, it has also been discovered that syntheticcyclodextrins may be used as host molecules, with a variety of mutantαHLs as sensor elements, to effect ion selectivity of the transmembranepore. For example, it has been discovered that the charge selectivity ofstaphylococcal α-hemolysin (αHL) may be manipulated by using twodifferent cyclodextrins as non-covalent molecular adapters.Anion-selective versions of αHL, including the wild-type pore andvarious mutants, become more anion-selective when β-cyclodextrin (βCD)is lodged within the channel lumen. By contrast, the negatively chargedadapter, heptakis-6-sulfato-β-cyclodextrin (s₇βCD), produces cationselectivity. The cyclodextrin adapters have similar effects when placedin cation-selective mutant αHL pores.

Most probably, hydrated Cl⁻ ions partition into the central cavity of CDmore readily than K⁺ ions, while s₇βCD introduces a charged ring nearthe midpoint of the channel lumen and confers cation selectivity throughelectrostatic interactions. The molecular adapters generate permeabilityratios (P_(K+)/P_(Cl−)) over a 200-fold range and may be used in the denovo design of membrane channels both for basic studies of ionpermeation and for applications in biotechnology.

As noted, the properties of transmembrane channels and pores may bemanipulated by cyclic oligosaccharides comprising glucose units(cyclodextrins) which act as molecular adapters for the pore formed bystaphylococcal α-hemolysin. Molecules of up to ˜2000 Da can betransported through a wide channel in the pore that is centered on themolecular seven-fold axis. Measurements of ionic currents indicate aweak anion selectivity (Menestrina, G., J. Membrane Biol., 90:177-190,1986).

Cyclodextrins reduce the conductance of the pore by lodging at a pointabout halfway through the channel, where the diameter is at itsnarrowest (−14 Å) (Gu, L.-Q., et al., Nature, 398:686-690, 1999).Further, channel blockers can bind to a cyclodextrin while it is in thechannel. For example, β-cyclodextrin (βCD) reduces the conductance of WT(wild-type) αHL from 658 pS to 240 pS in 1 M NaCl, pH 7.5, and a largevariety of organic molecules cause transient channel blockades bybinding within the WT-αHL-βCD complex (Gu, L.-Q., et al., Nature,398:686-690, 1999). The results with channel blockers suggest that asubstantial fraction of the ionic current flows through the center ofthe cyclodextrin molecule when it is lodged in the channel lumen.

Surprisingly, it has been discovered that molecular adapters, such ascyclodextrins, can be used to change the charge or ion selectivity of atransmembrane pore, such as an αHL pore. As demonstrated in Examples9-11, cyclodextrin adapters were shown to produce substantial changes inthe charge selectivity of αHL. For example, the WT-αHL pore equippedwith an anionic adapter, s₇βCD, was strongly cation selective(P_(K+)/P_(Cl−)=10), while a mutant αHL equipped with unmodified βCD wasstrongly anion selective (P_(K+)/P_(Cl−)=0.05). The direction of thesalt gradient had a significant effect on P_(K+)/P_(Cl−) values (Table2, below), which may be attributed to several causes, includingdifferential screening of charged groups, effects on the conformation ofthe protein and the onset of multi-ion transport conditions.Nevertheless, the general trends are unaffected by these deviations. Thecyclodextrin adapters produced no significant discrimination betweencations, as manifest in P_(K+)/P_(Na+) values.

Ion permeation remains one of the most disputatious areas of theoreticalbiophysics (Andersen, O. S., J. Gen. Physiol., 113:763-764, 1999;Normer, W., et al., J. Gen. Physiol., 113:773-782, 1999; Miller, C., J.Gen. Physiol., 113:783-787, 1999; Levitt, D. G., J. Gen. Physiol.,113:789-794, 1999). Computational demands place an understanding ofpermeation beyond present-day exact molecular dynamics simulations(Jakobsson, E., Methods, 14:342-351, 1998; Levitt, D. G., J. Gen.Physiol., 113:789-794, 1999). The two major practicable approaches,chemical kinetics and diffusion theory, are rich in adjustableparameters and it is not surprising they “explain” most experimentalobservations. For example, a permeability ratio of ten amounts to abarrier difference of only 1.4 kcal mol⁻¹, which is readilyaccommodated. Here is an example where the ability to measure farexceeds the ability to compute. Nonetheless, the findings herein areconsistent with qualitative notions about selectivity.

Ion selectivity clearly depends to a large extent on the dimensions of apore and the spatial distribution of charges at the entrance to andwithin the channel lumen (Hille, B. (1991) Ionic Channels of ExcitableMembranes (Sinauer, Sunderland, Mass.); Green, W. N., et al., Ann. Rev.Physiol., 53:341-359, 1991; Roux, B., et al., Science, 285:100-102,1999; Lear, J. D., et al., J. Am. Chem. Soc., 119:3212-3217, 1997;Kienker, P. K., et al., Biophys. J., 68:1347-1358, 1995). A “wide pore”with a radius greater than the Debye length (˜10 Å in 100 mM salt, ˜3 Åat 1000 mM) generally shows weak selectivity because ions in transitinteract primarily with water and other ions, rather than with the wallof the lumen (Kienker, P. K., et al., Biophys. J., 68:1347-1358, 1995).In this case, ion selectivities roughly reflect the diffusioncoefficients of individual ions in solution. Narrow pores, such asvoltage-gated K channels (d=3 Å), Na channels (d=4 Å), and gramicidin A(d=4 Å) are at the opposite extreme and show not only high chargeselectivity, but substantial discrimination among ions of the samecharge. Here, high selectivity arises through dehydration of ions in thechannel lumen and coordination by preorganized functional groups in aselectivity filter (Hille, B. (1991) Ionic Channels of ExcitableMembranes (Sinauer, Sunderland, Mass.); Eisenman, G., et al., J. Membr.Biol., 76:197-225, 1983), which in the case of K channels are the oxygenatoms of backbone carbonyls (Roux, B., et al., Science, 285:100-102,1999; Doyle, D. A., et al., Science, 280:69-77, 1998). Between theextremes of wide and narrow channels, mid-sized channels, such as thenicotinic acetylcholine receptor and the anion-selective GABAa receptorshow high charge selectivity, but low selectivity among ions of the samecharge. The selectivity of wide and mid-sized channels can be altered byusing mutagenesis to place or alter charged amino acid side chains alongthe conductive pathway and at its entrance (Saxena, K., et al.,Biochemistry, 38:2206-2212, 1999; Starostin, A. V., et al.,Biochemistry, 38:6144-6150, 1999; Kellenberger, S., et al., Proc. Natl.Acad. Sci. USA, 96:4170-4175, 1999; Kieckmann, G. R., et al., Biophys.J., 76:618-630, 1999)). Even for high selectivity channelselectrostatics can provide a prefilter mechanism (Roux, B., et al.,Science, 285:100-102, 1999; Doyle, D. A., et al., Science, 280:69-77,1998).

WT-αHL should be considered a “wide pore” (Menestrina, G., J. MembraneBiol., 90:177-190, 1986). The narrowest internal diameter is ˜14 Å nearMet-113, which is close to the cyclodextrin binding site (Gu, L.-Q., etal., Nature, 398:686-690, 1999; Song, L., et al., Science,274:1859-1865, 1996). In keeping with the assignment as a wide pore,WT-αHL is of high conductance (658 pS, 1 M NaCl, pH 7.5, −40 mV) and,despite the presence of charged residues throughout the channel lumen(FIG. 7 a), charge selectivity is weak (Menestrina, G., J. MembraneBiol., 90:177-190, 1986; under the conditions described hereinP_(K+)/P_(Cl−)=0.55-0.79).

The selectivity of αHL can be altered by introducing multiple chargedside chains into the channel lumen, as seen with αHL-CH1(P_(K+)/P_(Cl−)=5.1; 541 pS, 1 M KCl, pH 7.4, −40 mV), in which the netcharge of the lower half of the transmembrane barrel is changed from −7to −21. WT-αHL and αHL-CH1 pores both bound the neutral adapter βCD,which introduces a mid-sized constriction (internal diameter 6.2 Å(Jeffrey, G. A. & Saenger, W. (1991) in Hydrogen Bonding in BiologicalStructures, eds. Jeffrey, G. A. & Saenger, W. (Springer-Verlag, BerlinHeidelberg), pp. 309-350), still sufficient to admit a hydrated ion) asdemonstrated by the reductions in conductance: WT-αHL•βCD, g=240 pS, 1 MNaCl, pH 7.5, −40 mV; αHL-CH1•βCD, g=109 pS, 1 M KCl, pH 7.4, −40 mV.Both pores are anion selective when the adapter is bound. Therefore, theβCD adapter dominates ion selection as judged by the similar outcomes inboth an anion-selective and cation-selective backgrounds (Table 2). γCD,which contains eight glucose units, rather than the seven of βCD, isalso uncharged, with a larger internal diameter of 7.9 Å (Jeffrey, G. A.& Saenger, W. (1991) in Hydrogen Bonding in Biological Structures, eds.Jeffrey, G. A. & Saenger, W. (Springer-Verlag, Berlin Heidelberg), pp.309-350). As expected, γCD has a lesser effect than βCD on theconductance of the WT-AHL pore (WT-αHL•γCD: g=328 pS, 1 M NaCl, pH 7.5,−40 mV). The influence on anion selectivity was also reduced(P_(K+)/P_(Cl−)=0.38).

The seven sulfate groups of s₇βCD form a negatively charged ring (−7)(FIG. 7 b). This adapter greatly reduced the conductance of the WT-αHLpore: WT-αHL•s₇βCD, g=53 pS, 1 M NaCl, pH 7.5, 40 mV. Further, the porebecame cation selective, P_(K+)/P_(Cl−)=6.7-10. In this case,electrostatics predominate over the preference of the interior of thecyclodextrin for anions, in keeping with many studies in the literaturein which the introduction of charged rings at channel entrances or inthe lumen altered charge selectivity in a qualitatively predictablemanner. For example, the mitochondrial voltage-dependent anion channel(VDAC) was made cation selective by point mutagenesis (Blackly-Dyson,E., et al., Science, 247:1233-1236, 1990). In general, the placement ofnegatively charged side chains in the lumen favored cation selectivity,while positively charged side chains favored anion selectivity. Thevariation in permeability ratios was modest (P_(K+)/P_(Cl−) range0.6-1.9), as expected of a very high conductance channel (4.5 nS, 1 MKCl, +10 mV). In another case, the porin of Paracoccus denitrificans(g=2.6 nS, 1 M KCl, pH 6, +20 mV) was converted from a weakly anionicform (PK+/P_(Cl−)=0.35) to a highly cation selective pore(P_(K+)/P_(Cl−)=14) by two Arg->Glu mutations located along the barrelwall at the channel restriction (Saxena, K., et al., Biochemistry,38:2206-2212, 1999). In alamethicin, substitution of a neutral Gln inthe channel lumen near the C-terminal entrance with Lys, resulting insix additional charges per active hexamer, converted the cationselective channel (P_(K+)/P_(Cl−)=4.3) to an anion selective form(P_(K+)/P_(Cl−)=0.27). In all these cases, the lack of athree-dimensional structure does not permit the precise localization ofthe charged side chains.

Modified cyclodextrins have been used directly as ion channels. Forexample, Tabushi and colleagues made a β-cyclodextrin carryinghydrophobic chains on four of the seven 6-positions (Tabushi, I., etal., Tetrahedron Lett., 23:4601-4604, 1982). It was claimed that two ofthese molecule form a transmembrane pore. In a more extensive study,Lehn and collaborators made a “bouquet” molecule from β-cyclodextrin byattaching seven PEG chains to each face. This molecule showed some ofthe properties expected of a transmembrane pore (Pregel, M. J., et al.,Angew. Chem. Int. Ed. Engl., 31:1637-1639, 1992; Pregel, M. J., et al.,J. Chem. Soc. Perkin Trans., 2:417-426, 1995). In another study, acondensed monolayer of β-cyclodextrin with all seven 6-positionsmodified with long alkyl chains was deposited on a graphite electrode.The modified surface was permeant to the electroactive marker p-quinone(Odashima, K., et al., Analyt. Chem., 65:927-936, 1993). Transportthrough the central cavity was invoked because it could be blocked withguest molecules. Unfortunately, in none of these cases were clear-cutmeasurements of charge selectivity made.

In sum, the ion selectivity of a transmembrane pore may be modulatedusing the novel methods disclosed. The adapters can be regarded as crudemodular selectivity filters. By reducing the dimensions of the channellumen from “wide” to “mid-sized,” the adapters dominate the chargeselectivity of the pore. Unmodified βCD has an affinity for anions,while the negatively charged s₇βCD rejects anions, allowing cations topass in preference. Like site-directed mutagenesis, the adapter approachis versatile because various adapters can be used to program the sameprotein. Furthermore, mutagenesis and the adapter approach can becombined, for example, to increase the dwell time of the adapter on theprotein (Gu, L.-Q., et al., Nature, 398:686-690, 1999).

Protein pores with adapters are useful model systems with which to studythe details of ion permeation. In the case of the αHL•βCD system, theprotein is of known structure and both the protein and the adapter haveseven-fold symmetry. The adapter is not likely to produce any majorrearrangements of the protein. A similar approach may be used to alterthe activity of other proteins, e.g., to modify the active site of anenzyme. Greater pore selectivity may be achieved, for example, by usingan adapter with a ring of carbonyl groups similar to those found in theionophore valinomycin (Duax, W. L., et al., Biopolymers, 40:141-155,1996) or eukaryotic potassium channels (Doyle, D. A., et al., Science,280:69-77, 1998). The control of ion selectivity as disclosed herein maybe used in numerous aspects of biotechnology, including drug deliveryand biosensor design.

The following examples are offered to illustrate embodiments of theinvention, and should not be viewed as limiting the scope of theinvention.

EXAMPLES Example 1 Methods

Unless otherwise indicated, the following methods were used in Examples2-6.

Pore formation. Heptameric WT (wild-type) αHL formed by treatingmonomeric αHL purified from Staphylococcus aureus with deoxycholate(Bhakdi, S., et al., Proc. Natl. Acad. Sci. USA 78:5475-5479, 1981;Walker, B. J., et al., J. Biol. Chem. 267:10902-10909, 1992) wasisolated from SDS-polyacrylamide gels as described previously (Braha,O., et al., Chem. Biol. 4:497-505, 1997).

Planar bilayer recordings. A bilayer of1,2-diphytanoylphosphatidylcholine (Avanti Polar lipids, Birmingham,Ala., USA) was formed on a 100 to 150 μm orifice in a 25 μm thick teflonfilm (Goodfellow Corporation, Malvern, Pa., USA) separating twocompartments (2 ml each) of a planar bilayer apparatus (Montal, M., etal., Proc. Natl. Acad. Sci. USA 69:3561-3566, 1972). The solutions inthe compartments contained 1 M NaCl, 5 μM EDTA, 10 mM Na phosphate at pH7.5 in the cis chamber and pH 3.0 in the trans chamber. Heptameric αHL(0.5 to 1 μl at 0.05 to 0.2 ng/ml) was added to the cis compartment,which was stirred until a single channel inserted into the bilayer.Currents were recorded at a holding potential of 40 mV (cis at ground)by using a patch clamp amplifier (Axopatch 200B, Axon Instruments,Foster City, Calif.). The currents were low-pass filtered with abuilt-in 4-pole Bessel filter at 5 kHz and sampled at 20 kHz by computerwith a Digidata 1200 A/D converter (Axon Instruments). βCD (Aldrich) andthe analytes, ie., adamantane derivatives (Aldrich) were added to thetrans chamber.

Data analysis. Probabilities and mean durations were analyzed fromcurrent amplitude and dwell (residence) time histograms using pClamp 6.0(Axon Instruments) and are presented using Origin4.1 (Microcal,Northampton, Mass., USA). Measurements are given as the mean±SD. P_(P),P_(P●C) and P_(P●C●A) were obtained from the amplitude histograms afterfitting the peaks to Gaussian functions.

Example 2 Detection of Single Analyte

FIG. 6 is an example of analyte identification by the carrier techniqueof the present invention. In this example, the analyteadamantane-1-carboxylic acid is detected by measuring the modulation ofthe current carried by a single wild-type α-hemolysin pore.

Planar bilayer recordings were made under the following conditions:Buffer 1 M NaCl, 5 μM EDTA, 10 μM MOPS, pH 7.5; potential, 40 mV;α-hemolysin added to the cis chamber; γ-cyclodextrin added to the transchamber, adamantane-1-carboxylic acid added to the trans chamber. FIG. 6a shows a single channel current in the absence of carrier(cyclodextrin) and analyte (adamantane-1-carboxylic acid); FIG. 6 bshows step reduction in the current caused by a single carrier (5 μM)binding event; FIG. 6 c shows analyte (20 μM) binds to the carrier onthe pore and modulates the current with a characteristicconcentration-dependent signature.

Example 3 Guests (Analytes) on Host Molecule (Adapter) Reduce SingleChannel Currents

This example demonstrates that guest molecules bound to the hostmolecule reduced single channel currents.

The results are depicted in FIG. 1, which shows bilayer recordingsdemonstrating the interaction of a single αHL pore with βCD and themodel analytes 2-adamantanamine (A₁) and 1-adamantanecarboxylic acid(A₂). All traces were recorded at −40 mV (cis at ground). The buffer was1 M NaCl, 10 mM Na phosphate and 5 μM EDTA at pH 7.0 (cis) and pH3.0(trans). αHL was added to the cis chamber and βCD and the adamantanederivatives to the trans chamber. FIG. 1 a shows a single αHL porecontinuously open, −31.5 pA (level 1). FIG. 1 b shows that βCD (20 μM,trans) produces transient partial blockades of the channel, −11.5 pA(level 2). FIG. 1 c shows that 2-adamantanamine (20 μM, trans) does notaffect the fully open channel (level 1), but produces an additionalblock of αHL●βCD, −5.7 pA (level 3). FIG. 1 d shows that1-adamantanecarboxylic acid (20 μM, trans) produces additionalblockades, 4.7 pA (level 4), of longer duration than those produced by2-adamantanamine (level 3).

2-adamantanamine (A₁, 80 μM trans) reduced the conductance of thepartially blocked channel to 126.5 pS (SD=0.5, n=7) with a residencetime (τ_(A1)) of 2.54 msec (SD=0.21), but had no effect on thecompletely open channel (FIG. 1 c). A second guest,1-adamantanecarboxylic acid (A₂, 20 μM trans), also reduced theconductance of the partially blocked channel, this time to 112.2 pS(SD=3.2, n=7) with a residence time (τ_(A2)) of 14.0 msec (SD=0.8). Theguests competed for the single binding site in the αHL●βCD complex, sothat events due to each could be monitored in a mixture (FIG. 1 d).

Example 4 Interaction of αHL●βCD with Various Substituted Adamantanes

Table 1 demonstrates the results of the αHL●βCD interaction with varioussubstituted adamantanes^(a).

TABLE 1 (I_(P·C·A), I_(P·C))/ Residence Analyte (I_(P·C·A), I_(P))^(b)time (τ)(ms)^(c) 1/K^(A) _(f) (μM)^(d) 1-adamantanecarboxylic 0.346 ±0.005 14.0 ± 0.79 7.53 ± 1.16 acid 2-adamantanamine 0.286 ± 0.004 2.54 ±0.21  98.6 ± 14.28 hydrochloride 1-adamantanamine 0.294 ± 0.005 1.42 ±0.14 109.4 ± 4.95  hydrochloride 1-adamantanecarbox- 0.333 ± 0.005 3.99± 0.18 34.2 ± 2.77 amide 1-adamantanemethanol 0.352 ± 0.003 12.2 ± 1.4715.5 ± 5.26 1-adamantaneethanol 0.348 ± 0.003 30.6 ± 2.42 3.33 ± 1.07N-(1-adamantyl)- 0.348 ± 0.003 7.32 ± 2.13 14.00 ± 2.24  acetamide

-   a. Buffer: 1 M NaCl, 10 mM Na phosphate and 5 μM EDTA at pH 7.5    (cis) and pH 3.0 (trans). Data was acquired at −40 mV for 2.5 min,    n≧3 for all entries.-   b. I_(P●C●A), I_(P●C) and I_(A) are the current amplitudes of αHL    with βCD and analyte bound, αHL with only βCD bound and αHL with    nothing bound.-   c. The residence time of the analyte on αHL●βCD was obtained by    exponential fitting of the dwell (residence)-time histogram.-   d. K^(A) _(ƒ)is the equilibrium formation constant for the binding    of analyte to αHL●βCD obtained as described in Example 7.

Example 5 Analyte Signals Provide Identifying and Quantitative Data

This example demonstrates that the signal from an analyte can be usednot only to identify the analyte but also to quantitate it.

Specifically, FIGS. 4 a-c illustrate the response of αHL●βCD atdifferent analyte concentrations. FIG. 4 a shows the αHL●βCD (level 2)to αHL●βCD●A₁ (level 3) transitions at various 2-adamantanamine (A₁)concentrations. Representative segments of traces are shown depictingentire βCD occupancy events at 0, 40, 160 and 240 μM 2-adamantanamine.The conditions were the same as discussed for Example 3 (FIG. 1), butwith 40 μM βCD. FIG. 4 b shows plots of P_(P●C●A)/P_(P●C) versus [A] for2-adamantanamine and 1-adamantanecarboxylic acid. The slope yields theformation K^(A) _(ƒ) for αHL●βCD●A from analyte and αHL●βCD. FIG. 4 cshows plots of [C●A] versus [C] [A] for 2-adamantanamine and1-adamantanecarboxylic acid. The slope yields the formation K^(A)_(ƒ)for βCD●A from analyte and βCD in solution.

As expected, the frequency of αHL●βCD occupancy by analyte increaseswith analyte concentration (FIG. 4 a). The residence time ofβCD●2-adamantanamine on αHL is greater than βCD itself (FIG. 4 a), whichis true for all βCD●A in this study.

Applying Eq. 10 (see Example 7), K^(A) _(ƒ) values were obtained fromthe slope of a linear plot (FIG. 4 b). K^(A) _(ƒ) for1-adamantanecarboxylic acid and 2-adamantanamine (at pH 3.0) were found-to be respectively 1.35±0.19×10⁵M⁻¹ and 1.03±0.15×10⁴ M-1 (mean±SD,n=7), corresponding to AG values of −7.0 kcal mol⁻¹ and −5.5 kcal mol⁻¹.

Example 6 Simultaneous Identification and Quantitation of Two Analytes

This example demonstrates the use of sensing using a single sensorelement to simultaneously identify and quantitate two or more analytes,only one of which can occupy the receptor at a given moment.

In a mixture, signals from different analytes are recognized by theircharacteristic extents of channel block and residence times (Table 1).To illustrate this with αHL and the βCD adapter, an experiment wasperformed in which 1-adamantanecarboxylic acid (A₂) was kept constant at20 μM, while 2-adamantanamine (A₁) was varied. Current amplitudehistograms (FIG. 5 a), which revealed P_(P), P_(P●C) and P_(P●C●Ai), andEq. 8 were used to generate the total concentrations of the twoanalytes, [A₁]₀ and [A₂]₀. The values obtained were in close agreementwith the actual concentrations in the mixtures (FIG. 5 b).

Specifically, FIGS. 5 a-b illustrate analysis of currents from binarysolutions of analytes. FIG. 5 a shows current amplitude histograms for asingle αHL pore in the presence of βCD and 2-adamantanamine (A₁) and1-adamantanecarboxylic acid (A₂). The data has been fitted to Gaussianfunctions. The peak from unoccupied αHL is not shown. The conditionswere the same as in Example 3, FIG. 1, but with 40 μM βCD. Theconcentrations of the analytes, A₁ and A₂ respectively, in μM were: al,0, 0; a2, 0, 20; a3, 80, 20; a4, 160, 20; a5, 240, 20; a6, 320, 20. Asdepicted in FIG. 5 b, the experimentally measured concentrations of A₁and A₂, determined from the data in FIG. 5 a, are plotted against theactual concentration of A₂.

Example 7 Kinetic Scheme and Related Calculations

The interactions between the αHL pore (P), the cyclodextrin adapter (C)and analyte molecules (A_(i)) may be modeled by the following kineticscheme (Scheme 1):

The binding of each analyte molecule (A₁, A₂, . . . , A_(n)) to thecyclodextrin in solution or the cyclodextrin-pore complex excludesanother. Therefore, $\begin{matrix}{K_{f}^{C} = {{k_{on}^{C}/k_{off}^{C}} = {P_{P \cdot C}/\left( {P_{P} \cdot \lbrack C\rbrack} \right)}}} & (1) \\{K_{f}^{A_{i}} = {{k_{on}^{A_{i}}/k_{off}^{A_{i}}} = {P_{P \cdot C \cdot A_{i}}/\left( {P_{P \cdot C} \cdot \left\lbrack A_{i} \right\rbrack} \right)}}} & (2) \\{K_{f}^{C \cdot A_{i}} = {{k_{on}^{C \cdot A_{i}}/k_{off}^{C \cdot A_{i}}} = {P_{P \cdot C \cdot A_{i}}/\left( {P_{P} \cdot \left\lbrack {C \cdot A_{i}} \right\rbrack} \right)}}} & (3) \\{K_{f}^{A_{i}^{\prime}} = {{k_{on}^{A_{i}^{\prime}}/k_{off}^{A_{i}^{\prime}}} = {\left\lbrack {C \cdot A_{i}} \right\rbrack/\left( {\lbrack C\rbrack \cdot \left\lbrack A_{i} \right\rbrack} \right)}}} & (4)\end{matrix}$where K^(C) _(ƒ), K^(A) _(ƒ), K^(C•A) _(ƒ)and K^(A′) _(ƒ)are equilibriumformation constants. P_(P), P_(P•C) and P_(P•C•A) are probabilities ofoccurrence of the corresponding states. [C], [A] and [C•A] refer toconcentrations of free cyclodextrin, free analyte and thecyclodextrin-analyte complex in solution.

The total concentration of each analyte [A_(i)]₀ is obtained by applyingEq. 4: $\quad\begin{matrix}\begin{matrix}{\left\lbrack A_{i} \right\rbrack_{0} = {\left\lbrack A_{i} \right\rbrack + \left\lbrack {C \cdot A_{i}} \right\rbrack}} \\{= {\left\lbrack A_{i} \right\rbrack + {{K_{f}^{A_{i}^{\prime}}\lbrack C\rbrack}\left\lbrack A_{i} \right\rbrack}}}\end{matrix} & (5)\end{matrix}$where [α]and [C] are determined by Eq. 2 and Eq. 1: $\begin{matrix}{\left\lbrack A_{i} \right\rbrack = {P_{P \cdot C \cdot A_{i}}/\left( {P_{P \cdot C} \cdot K_{f}^{A_{i}}} \right)}} & (6) \\{\lbrack C\rbrack = {P_{P \cdot C}/\left( {P_{P} \cdot K_{f}^{C}} \right)}} & (7)\end{matrix}$

Therefore, the total concentration of an analyte can be determined fromthe expression: $\begin{matrix}{\left\lbrack A_{i} \right\rbrack_{0} = {\frac{1}{K_{f}^{A_{i}}} \cdot \left( {\frac{1}{P_{P \cdot C}} + \frac{K_{f}^{A_{i}^{\prime}}}{P_{P} \cdot K_{f}^{C}}} \right) \cdot P_{P \cdot C \cdot A_{i}}}} & (8)\end{matrix}$

Equilibrium constants. Before using Eq. 8, it is necessary to obtain theequilibrium constants K^(C) _(ƒ), K^(A) _(ƒ) and K^(A′) _(ƒ) for eachanalyte. K^(C) _(ƒ) can be obtained by titrating an αHL pore with βCD.K^(C) _(ƒ) is the slope of the line obtained by plotting P_(P•C)/P_(P)versus [C] based on the rearranged form of Eq. 1: $\begin{matrix}{{P_{P \cdot C}/P_{P}} = {K_{f}^{C} \cdot \lbrack C\rbrack}} & (9)\end{matrix}$K^(A) _(ƒ) and K^(A′) _(ƒ) are obtained from the slopes of plots basedon rearranged Eq. 2 and Eq. 4: $\begin{matrix}{\frac{P_{P \cdot C \cdot A}}{P_{P \cdot C}} = {K_{f}^{A}\lbrack A\rbrack}} & (10) \\{\left\lbrack {C \cdot A} \right\rbrack = {{K_{f}^{A^{\prime}}\lbrack C\rbrack} \cdot \lbrack A\rbrack}} & (11)\end{matrix}$where [C•A], [A] and [C]•[A] can be measured by applying Eq. 7:$\begin{matrix}{\left\lbrack {C \cdot A} \right\rbrack = {{\lbrack C\rbrack_{0} - \lbrack C\rbrack} = {\lbrack C\rbrack_{0} - \frac{P_{P \cdot C}}{P_{P}K_{f}^{C}}}}} & (12) \\{\lbrack A\rbrack = {{\lbrack A\rbrack_{0} - \left\lbrack {C \cdot A} \right\rbrack} = {\lbrack A\rbrack_{0} - \lbrack C\rbrack_{0} + \frac{P_{P \cdot C}}{P_{P}K_{f}^{C}}}}} & (13) \\{{\lbrack C\rbrack \cdot \lbrack A\rbrack} = {\frac{P_{P \cdot C}}{P_{P}K_{f}^{C}}\left( {\lbrack A\rbrack_{0} - \lbrack C\rbrack_{0} + \frac{P_{P \cdot C}}{P_{P}K_{f}^{C}}} \right)}} & (14)\end{matrix}$where [C]₀ and [A]₀ are the total concentrations of carrier and analyterespectively.

Probability measurement. P_(P), P_(P•C) and P_(P•C•A) are obtained bydetermining the fractional Gaussian areas corresponding to the relevantstates from the amplitude histograms of single channel recording traces.For analytes whose characteristic conductance levels in amplitudehistograms are too close to each other to be distinguished, theprobability P_(P•C•A) can be measured as a fraction of the total bindingtime T_(i) of analyte i to the total record time T:P _(P•C•A) ₁ =T _(i) /T  (15)

Each analyte contributes an exponential component of the residence time(mean residence time is τ) to the total analyte residence (dwell) timedistribution F(t): $\begin{matrix}{{F(t)} = {\sum\quad{\frac{N_{i}}{\tau_{i}}{\mathbb{e}}^{- \frac{t}{\tau_{i}}}}}} & (16)\end{matrix}$where N_(i) is the fitting scale factor. The total binding time for eachanalyte is $\begin{matrix}{T_{i} = {{\int_{0}^{\infty}{\frac{N_{i}}{\tau_{i}}{te}^{- \frac{t}{\tau_{i}}}\quad{\mathbb{d}t}}} = {N_{i} \cdot \tau_{i}}}} & (17)\end{matrix}$

Sensitivity. The sensitivity of αHL sensor element to a specific analyteA is defined as the total analyte concentration [A]₀ at which$\begin{matrix}{\frac{P_{P \cdot C \cdot A}}{P_{P \cdot C}} = 1} & (18)\end{matrix}$when [C]₀→0, [A]=[A]₀. So in this case, the sensitivity (from Eq. 10) is$\begin{matrix}{\lbrack A\rbrack_{0} = {1/K_{f}^{A}}} & (19)\end{matrix}$

As will be clear to those of skill in the art, the above calculationsmay be performed in real time by a built-in microprocessor or any othersuitable processing device.

Example 8 Materials, Methods and Permeability Ratio Summary

The following reagents were used in Examples 9-11. β-cyclodextrin wasfrom Aldrich (Milwaukee, Wis.) and γ-cyclodextrin from ACROS (Geel,Belgium). Heptakis-6-sulfato-β-cyclodextrin (s₇βCD) was prepared asdescribed in Vincent, J. B., et al., Analyt. Chem., 69:4419-4428, 1997.Buffers for planar bilayer recordings contained various concentrationsof KCl or NaCl and 10 mM K₂HPO₄ or Na₂HPO₄ (Sigma, St. Louis, Mo.) indeionized water (Millipore Corp., Bedford, Mass.), and were titrated topH 7.5 with 391 M HCl (EM Science, Gibbstown, N.J.). Experiments withthe mutant αHL-CH1 were done in KCl containing 10 mM K phosphate buffer,pH 7.4, and 5 μM EDTA.

The following proteins were used in Examples 9-11. The mutant αHL genesM113N, M113N/L135N and E111N/K147N were prepared by cassette mutagenesisin the plasmid αHL-RL2 (Cheley, S., et al., Protein Sci., 8:1257-1267,1999). These constructs contain the following additional changes overWT-αHL: Lys-8->Ala, Val-124->Leu, Gly-130->Ser, Asn-139->Gln,Ile-142->Leu. αHL polypeptides with these mutations behave similarly toWT-AHL in hemolysis assays and in planar bilayer recordings, at the saltconcentrations used herein (Cheley, S., et al., Protein Sci.,8:1257-1267, 1999). αHL-CH1 is one of several chimeric proteins thatfeature a transmembrane domain derived from the protective antigen ofanthrax toxin fused to the cap domain of αHL (laboratories of R. J.Collier and H. B., in preparation). Residues 119-140 inclusive of αHL(21 residues) were replaced with 22 residues 302-323 from protectiveantigen. The register of the β strands in the transmembrane domain isthat given by Petosa and colleagues (Petosa, C., et al., Nature,385:833-838, 1997).

Heptameric WT-αHL was formed by treating monomeric αHL, purified fromStaphylococcus aureus, with deoxycholate (Bhakdi, S., et al., Proc.Natl. Acad. Sci. USA, 78:5475-5479, 1981; Walker, B. J., et al., J.Biol. Chem., 267:10902-10909, 1992) and isolated from SDS-polyacrylamidegels as described (Braha, O., et al., Chem. Biol., 4:497-505, 1997).Mutant αHL polypeptides were prepared by coupled in vitro transcriptionand translation, with an S30 extract from Escherichia coli (no. L114A,Promega, Madison, Wis.) (Cheley, S., et al., Protein Sci., 8:1257-1267,1999). Heptamers were prepared from the mutants by assembly on rabbitred cell membranes, followed by preparative SDS-polyacrylamide gelelectrophoresis (Cheley, S., et al., Protein Sci., 8:1257-1267, 1999).

FIGS. 7 a-d depict representations of the proteins and cyclodextrinsused in Examples 9-11. FIG. 7 a is a sagittal section through the WT-αHLpore showing the location of all the charged side chains in the channellumen and the key hydrophobic residues M113 and L135. The site of thejunction in the chimera αHL-CH1 is indicated. FIG. 7 b shows thestructures of the β-cyclodextrins used. βCD, R═OH; s₇βCD, R═—OSO₃—. FIG.7 c is a schematic of the WT-αHL pore used showing βCD lodged in thelumen of the channel. The location is based on mutagenesis data (Gu,L.-Q. et al., Nature 398:686-690, 1999). FIG. 7 d shows sequences of thetransmembrane β barrels in WT-AHL (left) and αHL-CH1 (right).

Bilayer recordings were obtained as follows in Examples 9-11. A25-μm-thick teflon film (Goodfellow, Malvern, Mass.) with a 100-150 μmdiameter orifice was used as the partition between the two chambers (2ml each) of a teflon bilayer apparatus. The orifice was pretreated with1:10 hexadecane (Aldrich, Milwaukee, Wis.) Ipentane (Burdick & Jackson,Muskegon, Mich.). A solvent-free planar lipid bilayer of1,2-diphytanoyl-sn-glycero-phosphatidylcholine (Avanti Polar Lipids,Alabaster, Ala.) was formed covering the orifice (Montal, M., et al.,Proc. Natl. Acad. Sci. USA, 69:3561-3566, 1972; Hanke, W. & Schlue,W.-R. (1993) Planar Lipid Bilayers (Academic Press, London)). Apotential was applied across the bilayer with Ag/AgCl electrodes with1.5% agarose (Ultra Pure DNA Grade, Bio-Rad Laboratories, Hercules,Calif.) bridges containing 3 M KCl. Protein was added to the cischamber, which was at ground.

A positive potential indicates a higher potential in the trans chamberand a positive current is one in which cations flow from the trans tothe cis chamber. Single channel currents were recorded with an Axopatch200B patch-clamp amplifier (Axon Instruments, Inc., Foster City, Calif.)in the whole cell (β=1) mode with a CV-203BU headstage and filtered at 5kHz with a built-in 4-pole low-pass Bessel Filter. The data were eitheracquired by computer by using a Digidata 1200 A/D converted (Axon) orstored on DAT tape with a Dagan Unitrade DAS 75 recorder andsubsequently transferred to the computer after filtering at 1 kHzthrough a low-pass 8-pole Bessel filter (Model 900, Frequency Devices,Haverhill, Mass.). The data were acquired by using Clampex 7.0 software(Axon) after sampling at a rate of 20 kHz and analyzed with pClamp 6.03(Axon) and Origin (Microcal Software Inc., Northampton, Mass.) software.

Experiments were initiated by the addition of heptameric αHL to the ciscompartment with stirring until a single channel inserted into thebilayer. For the WT heptamer, oligomerized with deoxycholate, the finalconcentration was 3-30 ng ml⁻¹. For the mutant heptamers, oligomerizedon red cell membranes, the final concentration was ˜0.2 ng ml⁻¹. βCD ors₇βCD was added to the trans chamber to 40 μM. Experiments were at 22±2°C.

Data was analyzed in Examples 9-11 as follows. Single-channelconductances were determined by fitting the peaks in amplitudehistograms to Gaussian functions. The permeability ratios(P_(K+)/P_(Cl−), P_(K+)/P_(Na+)) were calculated from reversalpotentials by using the GHK equation. $\begin{matrix}{{\frac{P_{K^{+}}}{P_{{Cl}^{-}}} = {\frac{\left\lbrack a_{{Cl}^{-}} \right\rbrack_{t} - {\left\lbrack a_{{Cl}^{-}} \right\rbrack_{c}{\mathbb{e}}^{V_{r}{F/{RT}}}}}{{\left\lbrack a_{K^{+}} \right\rbrack_{t}{\mathbb{e}}^{V_{r}{F/{RT}}}} - \left\lbrack a_{K^{+}} \right\rbrack_{c}}\quad{under}\quad{asymmetrical}\quad{conditons}}},} & (20) \\{\frac{P_{K^{+}}}{P_{{Na}^{+}}} = {\frac{\left\lbrack a_{{Na}^{+}} \right\rbrack_{c}}{\left\lbrack a_{K^{+}} \right\rbrack_{t}}{\mathbb{e}}^{{- V_{r}}{F/{RT}}}\quad{under}\quad{biionic}\quad{{conditions}.}}} & (21)\end{matrix}$where V_(τ) is the reversal potential (i.e., the electrical potentialgiving zero current), ax is the activity of ion X (Zemaitis, J. F.,Clark, D. M., Rafal, M., & Scriver, N. (1986) Handbook of AqueousElectrolyte Thermodynamics: Theory and Application (American Instituteof Chemical Engineers, New York, N.Y.)), subscripts c and t representthe cis and trans compartments, and the other symbols have their usualmeanings. Vr was obtained by a polynomial fit of the current-voltage(I-V) data near zero current. For asymmetrical conditions, one chamber(cis or trans) contained 1000 mM KCl, while the other chamber contained200 mM KCl, except for the αHL-CH1 pore where: cis, 1000 mM KCl; trans,100 mM KCl. For biionic conditions, one chamber contained 1000 mM NaCland the other 1000 mM KCl. After the measurements, the membrane wasbroken to determine the contribution of electrode junction potentials.This value was normally smaller than 0.5 mV. Permeability ratios for αHLdepend upon several variables including pH and bilayer composition(Krasilnikov. O. V., et al., J. Membrane Biol., 156:157-172, 1997) andthe results obtained are valid only for the conditions stated.

Charge selectivity data is summarized in FIG. 10. For cation selectivepores (P_(K+)/P_(Cl−)) is shown (dark bars). For anion selective pores,the ratios in Table 2 have been inverted to give P_(Cl−)/P_(K+) (shadedbars).

Table 2 provides a summary of the permeability ratios (P_(K+)/P_(Cl−))and conductance values (g) determined in Examples 9-11 for the variousα-hemolysins, with and without adapters, that were evaluated.

TABLE 2 Minimum αHL- Charge diameter WT-αHL αHL-M113N E111N/K147NαHL-CH1 Adapter (e) (A) P_(K+)/P_(Cl−) g^(a)(pS) P_(K+)/P_(Cl−)g^(a)(pS) P_(K+)/P_(Cl−) g^(a)(pS) P_(K+)/P_(Cl−) g^(a)(pS) None 14 0.55± 0.02^(c) 658 ± 11 0.68 ± 0.03^(c) 622 ± 9 1.2 ± 0.1^(d) 634 ± 12 5.1 ±0.2^(e) 541 ± 11 0.79 ± 0.02^(d) 0.87 ± 0.04^(c) βCD 0 6.2 0.25 ±0.01^(c) 240 ± 5  0.079 ± 0.005^(c) 261 ± 4 0.15 ± 0.02^(d) 207 ± 7 0.82 ± 0.01^(e) 109 ± 9  0.23 ± 0.01^(d) 0.046 ± 0.006^(d) ₅₇βCD −7 <6.26.7 ± 0.4^(c) 53 ± 6 4.1 ± 0.2^(c)  40 ± 1 n.d n.d n.d  n.d.  10 ±0.2^(d)  6.1 ± 0.14^(d) _(r)CD 0 7.9 0.38 ± 0.04^(d) 328 ± 4  n.d. 287 ±5 n.d 275 ± 4 n.d. n.d.^(a)−40 mV, 1M NaCl, 10 mM Na phosphate, pH7.5; ^(b)−40 mV, 1MKC1, 10 mMK phosphate, pH7.4; ^(c) pH7.5, KCl in mM [cis/trans] was [200/1000];^(d) pH7.5, KCl in mM [cis/trans] was [1000/200]; ^(c) pH7.4, KCl mM[cis/trans] was [1000/100]. For each entry, three or more separateexperiments were performed and data acquired for at least 1 min. wasanalyzed. Permeability ratios are quoted as the mean±s.d.

Example 9 Cyclodextrins act as molecular adapters to enhance the anionselectivity of αHL pores

Various α-hemolysins and cyclodextrins were used in this example andthose which follow (FIG. 7).

As noted, βCD lodges transiently in the lumen of the WT-αHL pore, whereit acts as a non-covalent molecular adapter reducing the unitaryconductance (Table 2) and serving as a binding site for channel blockers(Gu, L.-Q., et al., Nature, 398:686-690, 1999). The homoheptamericWT-AHL pore is weakly anion selective (Menestrina, G., J. MembraneBiol., 90:177-190, 1986).

To determine whether the selectivity is altered while βCD is in thechannel lumen, single channel currents were recorded under asymmetricconditions: 1000 mM KCl cis, 200 mM KCl trans, pH7.5 (FIG. 8 a).Specifically, FIG. 8 a shows current recording in the presence of 40 μMβCD added to the trans side of the bilayer. The chambers contained 10 mMK phosphate, pH 7.5, with: cis: 1000 mM KCl; trans: 200 mM KCl. Thetransmembrane potential is indicated.

I-V curves were plotted for the contributions arising from theunmodified WT-αHL pore and the WT-αHL-βCD complex (FIG. 8 c).Specifically, FIG. 8 c shows I-V curves for αHL (□), αHL•βCD (◯) andαHL•s₇βCD (●) based on recordings made with cis: 1000 mM KCl; trans: 200mM KCl. Reversal potentials (V_(τ)) are marked by arrows.

Experiments were also performed with the opposite KCl asymmetry: 200 mMKCl cis, 1000 mM KCl trans, pH7.5 (FIG. 8 d). Specifically, FIG. 8 dshows I-V curves for αHL (□), αHL•s₇βCD (●) based on recordings madewith cis: 200 mM KCl; trans: 1000 mM KCl. The charge selectivities underthe various conditions were then calculated from V_(τ) and Eq. 20. TheWT-αHL•βCD complex (P_(K+)/P_(Cl−))=0.23-0.25) is significantly moreanion selective than the unmodified WT-αHL pore(P_(K+)/P_(Cl−))=0.55-0.79) (Table 2).

In addition, the effect of βCD on the mutant M113N was examined. M113Nbinds the cyclodextrin far more tightly than WT-AHL (Gu, L.-Q., et al.,Nature, 398:686-690, 1999). The almost non-selective M113N pore(P_(K+)/P_(Cl−))=(0.68-0.87) became highly anion selective with βCDbound (P_(K+)/P_(Cl−))=(0.046-0.079) (Table 2). The mutant M113N/L135N(Gu, L.-Q., et al., Nature, 398:686-690, 1999) gave similar results. Theeffect of γ-cyclodextrin, γCD, which contains eight glucose units, onthe selectivity of the WT-αHL pore was also tested. γCD enhanced theanion selectivity of the pore (P_(K+)/P_(Cl−)=0.38), but to a lesserextent than βCD (PK+/PcI—)=(0.23-0.25) (Table 2).

Example 10 The Anionic Adapter, Heptakis-6-Sulfato-β-Cyclodextrin(s₇βCD), Creates a Cation-Selective αHL Pore

In this example, the effects of a charged adapter on the ion selectivityof αHL were tested. Many commercially available derivatives of βCD arecomplex mixtures of regioisomers with different extents of substitution.Therefore, a highly purified preparation ofheptakis-6-sulfato-β-cyclodextrin (s₇βCD, FIG. 7) was tested.

s₇βCD bound to the WT-αHL pore from the trans side of the membraneproduced a substantial single channel block (FIG. 8 b; Table 2).Specifically, FIG. 8 b shows current recording in the presence of 40 μMs₇βCD added to the trans side. The chambers contained 10 mM K phosphate,pH 7.5, with: cis: 1000 mM KCl; trans, 200 mM KCl. The dwell time ofs₇βCD at pH 7.5 (τ=846±37 msec at −40 mV, n=3) was far greater than βCD(τ=0.84 msec±0.09 at −40 mV, n=8) and, as expected, it wasvoltage-dependent.

I-V curves were constructed for currents recorded under both cis/transand trans/cis KCl gradients (FIGS. 8 c, d) and charge selectivitiescalculated from V_(τ). Specifically, FIG. 8 c shows I-V curves for αHL(□), αHL•βCD (◯) and αHL•s₇βCD (●) based on recordings made with cis:1000 mM KCl; trans: 200 mM KCl. Reversal potentials (V_(τ)) are markedby arrows; FIG. 8 d shows I-V curves for αHL (□), αHL•s₇βCD (●) based onrecordings made with cis: 200 mM KCl; trans: 1000 mM KCl. TheWT-αHL•s₇βCD complex is strongly cation selective(P_(K+)/P_(Cl−)=6.7-10) (Table 2). αHL-M 113 N also became cationselective with s₇βCD bound (P_(K+)/P_(Cl−)=4.1-6.1) (Table 2).

The preference of WT-αHL•s₇βCD for Na⁺ and K⁺ was also compared. I-Vdata were recorded under biionic conditions with 1000 mM NaCl in onechamber and 1000 mM KCl in the other (FIG. 8 e). FIG. 8 e shows I-Vcurves under biionic conditions in 10 mM phosphate buffer, pH 7.5: □ and▪, αHL and αHL-s₇βCD with cis: 1000 mM KCl; trans: 1000 mM NaCl; ◯ and●, αHL and αHL•s₇βCD with cis: 1000 mM NaCl; trans: 1000 mM KCl. Ionselectivities were calculated from V_(τ) and Eq. 21. The permeabilityratio, P_(K+)/P_(Na+)=0.92, was the same for both cis/trans andtrans/cis buffer gradients. The value for the unmodified WT-αHL poreunder the same conditions was P_(K+)/P_(Na+)=1.0.

Example 11 Cation-Selective Mutant Pores Become Anion Selective with βCDas an Adapter

Because the increase in anion selectivity observed when βCD was used asan adapter for the WT-αHL pore was modest, in this example it isdetermined whether βCD would produce anion selectivity in acation-selective pore. To this end, αHL-CH 1, a chimeric protein thatfeatures a transmembrane domain derived from the protective antigen ofanthrax toxin fused to the cap domain of αHL, was examined. The netcharge per subunit in the transmembrane barrel of homoheptameric αHL-CH1is −21, compared with −7 in the WT-AHL barrel, and it is cationselective. The altered barrel in αHL-CH1 retains the site near Met-113,where cyclodextrins are believed to bind (Gu, L.-Q., et al., Nature,398:686-690, 1999). Once again, permeability ratios were determined fromV, values (FIGS. 9 a, b).

Specifically, FIGS. 9 a-b are bilayer recordings and I-V curves showingmodulation of single channel currents through the αHL-CH 1 pore by βCD.FIG. 9 a shows current recording in the presence of 40 μM βCD added tothe trans side of the bilayer. The chambers contained 10 mM K phosphate,pH 7.4, containing 5 μM EDTA, with cis: 1000 mM KCl; trans: 100 mM KCl.FIG. 9 b shows I-V curves for αHL-CH1 (□) and αHL-CH1•βCD (◯) based onrecordings made with cis: 1000 mM KCl; trans: 100 mM KCl. The datapoints represent mean values (±s.d.) from three different experimentsfor αHL-CH1 and two experiments for αHL-CH1•βCD (Arrows, V_(τ)).

βCD indeed converted the cation selectivity of αHL-CH1(P_(K+)/P_(Cl−)=4.4, pH7.4) to weak anion selectivity(P_(K+)/P_(Cl−)=0.8) (Table 2). The weakly cation selective mutantE111N/K147N (P_(K+)/P_(Cl−)=1.2) was also examined in the presence ofβCD, which converted it to an anion selective pore (P_(K+)/P_(Cl−)=0.15)(Table 2).

Other embodiments and uses of the invention will be apparent to thoseskilled in the art from consideration of the specification and practiceof the invention disclosed herein. All references cited herein,including all U.S. and foreign patents and patent applications, arespecifically and entirely hereby incorporated herein by reference,including, but not limited to, U.S. Provisional Patent Application Ser.No. 60/109,034 filed Nov. 18, 1998 and U.S. patent application Ser. No.09/122,583 filed Jul. 24, 1997. It is intended that the specificationand examples be considered exemplary only, with the true scope andspirit of the invention indicated by the following claims.

1. A method for sensing at least one analyte in a sample comprising:interacting the sample with a biosensor, the biosensor comprising asensor element having a receptor site and a host molecule, wherein thehost molecule interacts with the receptor site of the sensor element andthe analyte as an adapter between the analyte and the receptor site sothat the sensor element directly produces a detectable signal; anddetecting the signal; wherein detection of the signal indicates thepresence of said at least one analyte.
 2. A method for sensing at leastone analyte in a sample comprising: interacting the sample with abiosensor, the biosensor comprising a sensor element having a receptorsite and a host molecule, wherein the host molecule interacts with thereceptor site of the sensor element and the analyte as a carrier todeliver the analyte to the receptor site so that the sensor elementdirectly produces a detectable signal; and detecting the signal; whereindetection of the signal indicates the presence of said at least oneanalyte.
 3. The method of any one of claim 1 or 2 wherein interactingcomprises stochastic sensing.
 4. The method of any one of claim 1 or 2wherein the host molecule is non-covalently attached to the receptorsite.
 5. The method of any one of claim 1 or 2 wherein the host moleculeis covalently attached to the receptor site.
 6. The method of any one ofclaim 1 or 2 wherein the biosensor further comprises a bilayer and thesensor element comprises a channel disposed in the bilayer and coupledto the receptor site in manner that allows interaction of the hostmolecule with the receptor site to produce a signal using the channel.7. The method of any one of claim 1 or 2 wherein the biosensor furthercomprises a bilayer apparatus, the bilayer apparatus comprising abilayer separating the bilayer apparatus into a first compartment and asecond compartment and wherein the sensor element is disposed in thebilayer so that it forms a channel in the bilayer.
 8. The method ofclaim 7 wherein the sensor element is disposed in the first compartmentso that it forms a channel in the bilayer and the host molecule isdisposed in the second compartment.
 9. The method of claim 7 wherein thesensor element is disposed in the first compartment so that it forms achannel in the bilayer and the host molecule is disposed in the firstcompartment, the second compartment or both compartments.
 10. The methodof any one of claim 1 or 2 wherein the biosensor further comprises abilayer apparatus, the bilayer apparatus comprising a bilayer separatingthe bilayer apparatus into a first compartment and a second compartmentand wherein the sensor element is added to the first compartment andstirred to form a channel in the bilayer, and the step of interactingthe sample with the biosensor comprises adding the host molecule and thesample to the first compartment, the second compartment, or bothcompartments.
 11. The method of any one of claim 1 or 2 furthercomprising identifying the analyte using the signal.
 12. The method ofany one of claim 1 or 2 further comprising quantitating the analyteusing the signal.
 13. The method of any one of claim 1 or 2 wherein thehost molecule is a cyclodextrin.
 14. The method of claim 13 wherein thecyclodextrin is β-cyclodextrin (βCD).
 15. The method of claim 13 whereinthe cyclodextrin is s₇βCD.
 16. The method of any one of claim 1 or 2wherein the sensor element is a protein.
 17. The method of claim 16wherein the protein is selected from the group consisting of atransmembrane pore, an enzyme, an antibody and a receptor.
 18. Themethod of any one of claim 1 or 2 wherein the sensor element comprises apore.
 19. The method of claim 18 wherein the sensor element comprises agenetically engineered transmembrane protein pore.
 20. The method ofclaim 18 wherein the sensor element is an α-Hemolysin (HL) pore.
 21. Themethod of claim 20 wherein the sensor element is a wild-type α-Hemolysin(αHL) pore.
 22. The method of claim 20 wherein the sensor element is agenetically engineered or mutant α-Hemolysin (αHL) pore.
 23. The methodof any one claim 1 or 2 wherein the biosensor senses at least twoanalytes.
 24. The method of any one claim 1 or 2 wherein the signalcomprises a change in electrical current.
 25. The method of claim 24wherein the signal comprises a change in the magnitude and duration ofthe change in the current.
 26. The method of any one of claim 1 or 2wherein the analyte is an organic molecule.
 27. The method of any oneclaim 1 or 2 wherein the analyte is not charged.
 28. The method of anyone claim 1 or 2, wherein the signal is selected from the groupconsisting of a change in fluorescence, a change in electrical currentand a change in force.